Liposomes and lipid complexes have been long recognized as drug delivery systems which can improve therapeutic and diagnostic effectiveness of many bioactive agents and contrast agents. Experiments with a number of different antibiotics and X-ray contrast agents have shown that better therapeutic activity or better contrast with a higher level of safety can be achieved by encapsulating bioactive agents and contrast agents with liposomes or lipid complexes. Research on liposomes and lipid complexes as encapsulating systems for bioactive agents has revealed that a successful development and commercialization of such products requires reproducible methods of large scale production of lipid vesicles with suitable characteristics. Consequently, workers have searched for methods which consistently produce liposomes or lipid complexes of the required size and concentration, size distribution and, importantly, entrapping capacity, with flexible lipid composition requirements. Such methods seek to provide liposomes or lipid complexes with consistent active substance to lipid ratio while respecting currently accepted good manufacturing practices for pharmaceutical products.
Conventional liposome and lipid complex preparation methods include a number of steps in which the bilayer-forming components (for example, phospholipids or mixtures of phospholipids with other lipids e.g., cholesterol) are dissolved in a volatile organic solvent or solvent mixture in a round bottom flask followed by evaporation of the solvent under conditions, such as temperature and pressure, which will prevent phase separation. Upon solvent removal a dry lipid mixture, usually in form of a film deposit on the walls of the reactor, is hydrated with an aqueous medium which may contain dissolved buffers, salts, conditioning agents and an active substance to be entrapped. Liposomes or lipid complexes form in the hydration step such that a proportion of the aqueous medium becomes encapsulated in the liposomes. The hydration can be performed with or without energizing the solution by means of stirring, sonication or microfluidization or with subsequent extrusion through one or more filters, such as polycarbonate filters. The free non-encapsulated active substance can be separated for recovery and the product is filtered, sterilized, optionally lyophilized, and packaged.
Other methods of making liposomes or lipid complexes involving injection of organic solutions of lipids into an aqueous medium with continuous removal of solvent, use of spray drying, lyophilization, microemulsification and microfluidization, and the like have been proposed in a number of publications or patents. Such patents include, for example, U.S. Pat. No. 4,529,561 and U.S. Pat. No. 4,572,425.
Cisplatin—cis-diamine-dichloroplatinum (II)—is one of the more effective anti-tumor agents used in the systemic treatment of cancers. This chemotherapeutic drug is highly effective in the treatment of tumor models in laboratory animals and in human tumors, such as endometrial, bladder, ovarian and testicular neoplasms, as well as squamous cell carcinoma of the head and neck (Sur, et al., 1983 Oncology 40(5): 372-376; Steerenberg, et al., 1988 Cancer Chemother Pharmacol. 21(4): 299-307). Cisplatin is also used extensively in the treatment of lung carcinoma, both small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) (Schiller et al., 2001 Oncology 61(Suppl 1): 3-13). Other active platinum compounds (defined below) are useful in cancer treatment.
Like other cancer chemotherapeutic agents, active platinum compounds such as cisplatin are typically highly toxic. The main disadvantages of cisplatin are its extreme nephrotoxicity, which is the main dose-limiting factor, its rapid excretion via the kidneys, with a circulation half life of only a few minutes, and its strong affinity to plasma proteins (Freise, et al., 1982 Arch Int Pharmacodyn Ther. 258(2): 180-192).
Attempts to minimize the toxicity of active platinum compounds have included combination chemotherapy, synthesis of analogues (Prestayko et al., 1979 Cancer Treat Rev. 6(1): 17-39; Weiss, et al., 1993 Drugs. 46(3): 360-377), immunotherapy and entrapment in liposomes (Sur, et al., 1983; Weiss, et al., 1993). It has been reported that antineoplastic agents, including cisplatin, entrapped in liposomes have a reduced toxicity, relative to the agent in free form, while retaining antitumor activity (Steerenberg, et al., 1987; Weiss, et al., 1993).
Cisplatin, however, is difficult to efficiently entrap in liposomes or lipid complexes because of its low aqueous solubility, approximately 1.0 mg/mL at room temperature, and low lipophilicity, both of which properties contribute to a low cisplatin/lipid ratio.
Liposomes and lipid complexes containing cisplatin suffer from another problem—stability of the composition. In particular, maintenance of bioactive agent potency and retention of the bioactive agent in the liposome during storage are recognized problems (Freise, et al., 1982; Gondal, et al., 1993; Potkul, et al., 1991 Am J Obstet Gynecol. 164(2): 652-658; Steerenberg, et al., 1988; Weiss, et al., 1993) and a limited shelf life of liposomes containing cisplatin, on the order of several weeks at 4° C., has been reported (Gondal, et al., 1993 Eur J. Cancer. 29A(1): 1536-1542; Potkul, et al., 1991).